著者らは、訓練とキルシュナー鋼ラボカエル施設の使用、およびサポートのためのバン·バクター·ラボのメンバーのボブ·フリーマンに感謝したいと思います。我々は、 図1の画像のための光学顕微鏡での支援のためにハーバード大学医学部ニコンイメージングセンターに感謝します。この作業は、次の要素によって賄われていた：NRSA NIHのフェローシップとLALにNIHのK99交わり、ベーシックサイエンス·パートナーシップ資金（ <a href="https://bsp.med.harvard.edu/" target="_blank">https://bsp.med.harvard.edu/</a> AEF）、およびNIHのRO1 NS035909をDVVへ

注：詳細な情報と優れたプロトコルは、次の手順（ 例 8-12）に、より具体的に焦点を合わせることを他の場所で公開されているように、我々は、唯一の短い中で最初の2つのセクションの手順を説明します。さらに、生細胞培養におけるアフリカツメガエル脊髄ニューロンでの作業の一般的なプロトコルは、以前に詳細な方法の記事8に掲載されました。ここで我々は正常に実行し、ステップ3で、神経管の解剖とめっきプロトコルのトラブルシューティングを行うための十分な情報を提供していないが、我々は非常に、このビデオを補完するものとして、その記事を見直すことをお勧めします。 アフリカツメガエルの卵の1。 体外受精<ol><li>採卵前絨毛性ゴナドトロピン（400単位/カエル）12-18時間を注射した女性のカエルの卵を入手してください。 1X Marcの修正リンガー溶液（MMR）（0.1MのNaCl、2.0mMのKClを、1.0mMのMgSO 4を 、2.0 mMのCaCl 2に卵を集める、5.0mMのHEPES、pH7.4の9）。 </li><li>以前に9述べたように、みじん切りにした精巣を用いた in vitro で卵を受精させる。 </li><li>少なくとも20分後、3〜5分間1X MMRの2％システイン（NaOHでpHを7.8に持って来た）で胚を培養することによって胚ゼリーコートを脱ぐ。 3：0.1X、MMR（88のNaCl、1mMのKCl、0.7 mMのCaCl 2、1mMののMgSO 4、2.5mMの飽和NaHCO 3、5mMのHEPES、pH7.8）にまたは0.1XのMBS（修正バルトの生理食塩水、1Xレシピ）で洗う-5回、注射するまで室温で胚を保つ、または必要に応じて、14から18で行わ胚°Cの開発を遅らせるため。 </li></ol> 2。 RNAのマイクロインジェクション注：私達はここにRNAの注入を利用しながら、これらの技術は、RNAに限られるものではなく、DNA、タンパク質、または遺伝子操作のために修飾された核酸を使用することもできますし、以前12に記載されている。 <ol><li>細胞骨格やその他の構造arを標識するための実験、RNAに先立ちeはmMessage mMachineキット（Ambion）を用いて線形化されたDNA鋳型から転写された。 mRNAは、翻訳を促進し、胚の劣化を防止するために、5 &#39;キャップと3&#39;ポリアデニル化尾を必要とします。 PCS2 +は、この目的のために一般的に使用されるベクトルであり、転写キットは合成反応時のキャップアナログを含む。我々は、ヌクレアーゼフリー蒸留H 2 0で転写されたmRNAを再度サスペンド、500と2000の間にストック濃度でng /μLの、-80℃で保管プロンプトに続いて注射の前に、注射に適した濃度になるように蒸留H 2 0中のRNAを希釈します。わずかなボリュームが胚に注入されるように、バッファに再懸濁する必要はありません。私たちの最終的な注入濃度は、構造に応じて、通常は50から200 PG / NLであり、多くのラボは伝統の総容積の約1％である10 NL、最大注入するものの、我々は通常、胚あたり4 NLまで注入します胚。 RNAは高用量で毒性があること、および胚属あたりの線量ができます最大5 ngには、特定の遺伝子や純度に応じて許容することができるものの、リリー、10 pgのから1 ngの範囲である。しかし、イメージングタンパク質局在化のために、可能な限り低いレベルが過剰発現のアーチファクトを回避するために注入する必要があります。このプロトコルにおけるEB1-GFPのRNAが胚あたり250 pgの最終的な量で使用されています。 </li><li> 〜0.2μmの先端径に、ニードルプラー9キャピラリー引いて、注射針を用意します。熱644、プル125、速度70、時間250、しかし、これらのパラメータはマシンによって異なり、フィラメントを（私たちが現在2.5ミリメートルを使用し変更した後に再調整する必要があります。サッター、P-87プラーにより、我々は次の設定を使用ワイド2.5ミリメートル）である四角い箱フィラメント。顕微鏡下で、羽のような形状を生成するために角度を鉗子で針の先端を破る。そこに注射針を破壊し、充填のための他の方法（例11,12の場合）ですが、我々は小さなドロップ（0.5から1μl）を配置することにより、RNAと針を埋めるのバックエンドに針。胚にマイクロインジェクションするために、ピコインジェクター（メディカルシステムズ）、最も一般的な市販の注入システムの数、2、およびNanoject（ドラモンド·サイエンティフィック社は、）（詳細については11を参照）があります。我々は、一貫性のあるナノリットルボリュームを配信するために、時間のデジタル設定された期間のために圧縮ガスを使用するメディカルシステムズPLI-100ピコインジェクタを使用しています。注入量は、ステージのマイクロメーターを用いて、それぞれの新しいマイクロピペットに校正する必要があり、注入時間は、注入RNAの所望量を達成するために調整されるべきである。 </li><li> 1で受精した胚を置き - 4細胞期に0.1X MMRの5％フィコールを含むプラスチック皿に。鉗子で固定胚を保持、または保持プラットフォーム（塑像用又は歯科用のワックスと皿の底に付着した約1mmのグリッドを持つプラスチックメッシュ、、）に置くこと、（ステップ2.1を参照してください動物の割球に目的のボリュームを注入）注入量に関する詳細については。いくつかのLOCで配布（例えば、4細胞期胚のために、我々は通常、2細胞期のに対し、各割球で1〜2回注射するより均一な分布を得るために、動物の球のそれぞれに胚全体の管理ポイント、少なくとも1つの注入、胚、当社）は、各割球の2-4倍を注入します。 2から4細胞期まで待つことの利点は、胚が正常に切断するために始めていることを確認することです。時間が本質である場合は、あなたは注入される必要がある多くの胚以外の最終結果にはほとんど差が、1細胞期に開始することができます。古い段階の胚も同様に使用すると、特定の種​​類の細胞が注射を対象にしているなら、これは特に有用であるが、我々は一般的に、より幅広い表現を好むように、我々はより多数の注射の必要性を低減するために若い胚に注入することができます。 </li><li> 0.1X MMRを含むプラスチックシャーレに注入された胚を転送し、それらがステージ20から23 13まで開発することができます。 °Cの希望のSPEに応じて14から22に胚を培養する開発のED。 （翌日神経管の解剖については、後の日のために〜22℃を使用し、〜14℃を使用してください） </li></ol> 3。神経管の解剖とめっき<ol><li>前に解剖を行うには、14の培養皿を準備します。まず、コートマテックまたはLab-Tek製の培養皿のいずれかのカバーガラスの表面上にプールへは200μg/ PBS中ポリリジンの十分な解決策とカバーガラス、そして代わりに1時間（インキュベート、人は座っていたカバーガラスを使用することができますイメージングと免疫細胞化学のためのスライドガラス上の後に配置するため、シャーレ内の緩い）。吸引除去し、PBSで3回の過剰で洗浄し、乾燥させます。その後、10μgの/ PBS中ラミニンでコートの料理は37度で1時間（私たちは、通常十分な表面上のプールにカバースリップあたり500μlを、使用してください）​​。ラミニン液を吸引除去し、ラミニン被覆表面はエアインタフェースにさらされるものにならないように注意しながら、PBSの過剰で3回洗浄する。 REPLAC文化メディアと電子のPBS（50％リンゲル、49％L-15培地、1％ウシ胎児血清、プラス50μg/ mlのペニシリン/ストレプトマイシンおよびゲンタマイシン、pH7.4およびフィルター滅菌）。 （リンゲルメディア、115 mMのNaCl、2.5mMのKCl、2mMのCaCl 2、10mMのHepes緩衝液（pH7.4）、0.5mMのEDTA）。私たちのプロトコルはこのメディアを使用していますが、これは神経細胞がエネルギーを保有減少している年上のアフリカツメガエルの網膜神経節文化のための一般的な濃縮されたメディアであることに留意すべきである。ヤング脊髄文化が生き残り、さらなる成長因子なしで純粋なリンガーのメディア以上の24時間で成長し、これは実際にこのシステムを使用することの利点の一つであるでしょう。オプション：追加NT3とBDNFの軸索伸長を高めるために培地へ（25 ng /μLのそれぞれの最終濃度まで）。 </li><li>なぜなら注入mRNAの発現の変動により、胚は蛍光のモザイクを示すかもしれません。事前EXPそれら胚を識別するために解剖、蛍光の存在のためのスクリーンの胚を、実行することに神経管の蛍光融合タンパク質をRESS。 Steinberg社のメディア（58 mMのNaCl、0.67のKCl、0.44 mMののCa（NO 3）2、1.3mMのMgSO 4を 、4.6 mMトリス、次に7.8のpHでいっぱいアガロースコーティングされたプラスチック製の皿に20から23の段階で蛍光胚を置きオートクレーブ処理したもの）5。アガロースコーティングされた皿、0.1X、MMRとlet硬化の溶融1％アガロースとプラスチックの皿のコートボトムを作る。そのダメージはその後の解剖手順中に使用される微細鉗子及びタングステン針には発生しませんので、これはソフト面を提供する。ステージ23を超えた胚の郭清は可能ですが、組織がお互いにもっと密接に付着するので、コラゲナーゼによる長期の治療を必要とすることがより困難である。 </li><li>解剖スコープ]で、細かい鉗子で卵黄膜を除去した後、切開のシリーズを作ることによって、胚全体の背の部分を分離します。 1鉗子が所定の位置に胚を保持している間に、上の切開を作るために第二を使用中空内部を露出する胚の側。次に、神経管を含む背の部分を分離するために、それによって半分に胚を切断、胚の背側と腹側半分の間の組織に沿って挟まないように両方の鉗子を使用しています。 </li><li>組織を緩めるために、回転子上の15から20分間、Steinberg社のメディアで2 mg / mlのコラゲナーゼとエッペンドルフチュー​​ブに背側外植片を配置して、新鮮なSteinbergの溶液を用いてアガロースプラスチックでコーティングされた皿に背側外植片をピペット。あるいは、外植片は、全体の郭清はその後行うことができた15分間、コラゲナーゼ溶液を含むペトリ皿に配置することができます。 </li><li>ピンセットを使って、そっと背側と腹側表皮脊索から神経管を解剖。表皮と皮下組織の間に鉗子の先端をスライドさせて、徐々に神経管の下を明らかにし、表皮を引き戻す。その後、間の先端をスライドさせて組織を保持するために1つ鉗子を使用し、別の神経管や脊索。最後に、チューブの両側に体節を削除するには、鉗子を使用しています。 </li><li>ステップ3.1で説明したように、培地を充填したアガロースコーティングされた皿に神経管を転送します。 </li><li>いくつかの神経管を集めた後、広がって、媒体（ステップ3.1で調製した）を充填した後、尖ったタングステン針やピンセット、プレート培養皿のピースを使って多くの作品（約20、全体のチューブが分離されている場合）に切る行に均等に植片。 </li><li>これは取り付ける細胞を乱すため、めっき後、お皿を移動しないでください。 20から22の周りにメッキされた神経管の外植片をインキュベート℃に我々は通常、室温で一晩ベンチ上での細胞の皿を残す。 アフリカツメガエルの神経細胞の利点の一つは、CO 2レベルまたは温度を調節する細胞培養のための特別な保育器が必要とされていないことです。神経突起と成長円錐は室温12で観察し、画像化することができるめっき後-24時間、伸長が成長因子の添加により加速することができますが。一般的には、RNAまたはプラスミド産物の発現は、細胞間の変数式になるでしょう、そして、その結果、成長円錐の間の蛍光発現レベルの範囲があるかもしれません。我々は、これは特定の構築にもよるが、めっき後48時間を超えて存続するために蛍光タンパク質のRNA発現を観察した。 </li></ol> 4。代表的な結果 図1Aに要約されているプロトコルを実行した後、健康な、正しく解剖し、培養した神経外植片は、 図1Bに示すラミニン/ポリリジン基板上に、あらゆる方向に多数の軸索または神経突起を発送させていただきます。軸索先端の成長円錐は、LOを調べるために画像全体の運動力学に、 図1Cに見られるように、DIC光学、、、または高分解能蛍光顕微鏡法のいずれかによって結像させることができる蛍光タグを付けた細胞骨格タンパク質のcalizationは、例えば、EB1-GFPは、 図1Dに示されている。 <img alt="図1" src="/files/ftp_upload/4232/4232fig1.jpg" /> 図1：実験プロトコルと期待される成果の概要。 a）プロトコルのフローチャート。 3日目に解剖に関する詳細については、ムービーを参照してください。全体の神経管は、約20同じ大きさの片に切断し、カバースリップ上の行に均等に分散させる必要があります。この漫画では、はさみは、微細鉗子で組織を切断表現するために描かれている。外植片から出て成長中の軸索や神経突起のCG絨毛性ゴナドトロピンB）はDIC像（左上隅に植）。成長軸索の先端の成長円錐のC）の高倍率像。成長円錐におけるEB1-GFPのD）の蛍光顕微鏡画像。スケールバーは10μmである。 <a href="http://www.jove.com/files/ftp_upload/4232/4232fig1large.jpg" target="_blank">拡大図を表示するには、ここをクリックしてください</a> 。

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Neurosci. 8, 717-719 (2005).</li><li>Weinl, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+turning+of+Xenopus+retinal+axons+induced+by+ephrin-A5.">On the turning of Xenopus retinal axons induced by ephrin-A5.</a> Development. 130, 1635-1643 (2003).</li><li>Knoll, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stripe+assay+to+examine+axonal+guidance+and+cell+migration.">Stripe assay to examine axonal guidance and cell migration.</a> Nat. Protoc. 2, 1216-1224 (2007).</li><li>Wheeler, G. N., Hamilton, F. S., Hoppler, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inducible+gene+expression+in+transgenic+Xenopus+embryos.">Inducible gene expression in transgenic Xenopus embryos.</a> Curr. Biol. 10, 849-852 (2000).</li></ol>

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条件が適切であるかどうかをアフリカツメガエルの神経外植片は、ラミニン/ポリリジン基板上にメッキ後24時間で、非常に堅牢な方法で神経突起を出す。典型的な長さが100μm以上であるが、この基板では、成長円錐の運動性は非常にあり、外植片から外側に向かって、すべての方向に延びる、1ミリメートルまでの軸索の長さを実現することができます。神経突起が出て成長していない...

<table border="1"><tbody><tr><td> 試薬の名称 </td><td> 会社 </td><td> カタログ番号 </td></tr><tr><td>絨毛性ゴナドトロピン</td><td>アージェント化学研究所</td><td> C-HCG-ON-10 </td></tr><tr><td>システイン</td><td>シグマアルドリッチ</td><td> 52-90-4 </td></tr><tr><td> mMessage mMachineキット</td><td>アンビオン</td><td> AM1340 </td></tr><tr><td> Borosil針を1.2ミリメートル（外径）×0.9ミリメートル（ID）をキャピラリー</td><td> FHC </td><td> 30-31-0 </td></tr><tr><td>フィコール</td><td>シグマ</td><td> F2637 </td></tr><tr><td>デュモン第5 Biologieイノックス鉗子</td><td>ファイン科学ツール</td><td> 11252から20 </td></tr><tr><td>コラゲナーゼ</td><td>シグマアルドリッチ</td><td> 9001-12-1 </td></tr><tr><td>マテック料理</td><td>マットテック株式会社</td><td> P35G-1.5-14-C </td></tr><tr><td> L-15 </td><td>インビトロジェン</td><td> 21083-027 </td></tr><tr><td>ポリ-L-lysiね</td><td>シグマ</td><td> P-1399 </td></tr><tr><td>ラミニン</td><td>シグマ</td><td> L2020 </td></tr><tr><td> NT3 </td><td>シグマ</td><td> N1905 </td></tr><tr><td> BDNF </td><td>シグマ</td><td> B3795 </td></tr></tbody></table>

neural explant cultures from xenopus laevis

軸索ガイダンスの複雑なプロセスは、大きく成長している軸索の先端の動的運動性構造体である成長円錐によって駆動される。軸索伸長時には、成長円錐が前方成長円錐を推進し、正確にその具体的な目標1を見つけるために移動するために、その細胞骨格を調節するためのガイダンスキュー複数の情報源を統合する必要があります。この統合は、細胞骨格のレベルで起こり、まだ浮上していると成長円錐内の細胞骨格タンパク質およびエフェクターダイナミクスの検査は、これらのメカニズムの解明を可能にすることができる。 アフリカツメガエルの成長円錐は高い実行するのに十分（直径10〜30ミクロン）を大きくする方法解像度の細胞骨格の動態のライブイメージング（ 例 2-4）と他の脊椎動物に比べてラボ環境で隔離し、操作しやすい。カエルは、発達神経生物学研究、成長円錐のマイクプリアンプに重要な初期の洞察力のための古典的なモデルシステムであるrotubuleのダイナミクスは、当初このシステム5-7を使用して発見された。この方法8では、卵は収集され、 試験管内で受精し、遺伝子発現を操作するためにコードするRNA蛍光標識された細胞骨格融合タンパク質または他の構造物を注入し、その後神経管ステージに開発することができました。神経管は解剖によって分離され、その後培養し、神経突起outgrowingに成長円錐が結像される。本稿では、この方法では、その後の高解像度画像解析のための文化アフリカツメガエルの成長円錐になっているゴールを実行する方法について説明します。我々は+ TIPの融合タンパク質EB1-GFPの例を提供していますが、この方法は成長円錐内に自分の行動を明らかにするために、タンパク質の任意の数に適用することができます。

Watch this Scientific Journal Video about Neural Explant Cultures from Xenopus laevis at JoVE.com

Neural Explant Cultures from Xenopus laevis

解剖からの培養神経外植<em&gt;アフリカツメガエル</em&gt;蛍光融合タンパク質を発現する胚は成長円錐の細胞骨格ダイナミクスのイメージングを可能にします。

からの神経外植文化<em&gt;アフリカツメガエル</em

The complex process of axon guidance is largely driven by the growth cone, which is the dynamic motile structure at the tip of the growing axon. During ...

neural-explant-cultures-from-xenopus-laevis

Research

JoVE Journal

Neuroscience

Neural Explant Cultures from Xenopus laevis

12.5K ビュー.  Harvard Medical School. 解剖からの培養神経外植アフリカツメガエル蛍光融合タンパク質を発現する胚は成長円錐の細胞骨格ダイナミクスのイメージングを可能にします。

ビデオ: Neural Explant Cultures from Xenopus laevis

Neural-Colony Forming Cell Assay: An Assay To Discriminate Bona Fide Neural Stem Cells from Neural Progenitor Cells

The neurosphere assay (NSA) is one of the most frequently used methods to isolate, expand and also calculate the frequency of neural stem cells (NSCs). Furthermore, this serum-free culture system has also been employed to expand stem cells and determine their frequency from a variety of tumors and normal tissues. It has been shown recently that a one-to-one relationship does not exist between neurosphere formation and NSCs. This suggests that the NSA as currently applied, overestimates the frequency of NSCs in a mixed population of neural precursor cells isolated from both the embryonic and adult mammalian brain. This video practically demonstrates a novel collagen based semi- solid assay, the neural-colony forming cell assay (N-CFCA), which has the ability to discriminate stem from progenitor cells based on their long-term proliferative potential, and thus provides a method to enumerate NSC frequency. In the N-CFCA, colonies &ge;2 mm in diameter are derived from cells that meet all the functional criteria of a NSC, while colonies &lt; 2mm are derived from progenitors. The N-CFCA procedure can be used for cells prepared from different sources including primary and cultured adult or embryonic mouse CNS cells. Here we use cells prepared from passage one neurospheres generated from embryonic day 14 mice brain to perform N-CFCA. The cultures are replenished with proliferation medium every seven days for three weeks to allow the plated cells to exhibit their full proliferative potential and then the frequency of neural progenitor and bona fide neural stem cells is calculated respectively by counting the number of colonies that are &lt; 2mm and the ones that are &ge;2mm in reference to the number of cells that were initially plated.

This video protocol demonstrates how to discriminate and enumerate bona fide neural stem cells in a mixed population of neural precursor cells using the neural colony-forming cell assay.

神経コロニー形成細胞アッセイ：神経前駆細胞から善意の神経幹細胞を区別するアッセイ

The neurosphere assay (NSA) is one of the most frequently used methods to isolate, expand and also calculate the frequency of neural stem cells (NSCs). ...

神経科学

Isolation and Culture of Neural Crest Cells from Embryonic Murine Neural Tube

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to mesenchymal transition (EMT) and migrates throughout the embryo, giving rise to diverse cell types 1-3. NC also has the unique ability to influence the differentiation and maturation of target organs4-6. When explanted in vitro, NC progenitors undergo self-renewal, migrate and differentiate into a variety of tissue types including neurons, glia, smooth muscle cells, cartilage and bone. 
NC multipotency was first described from explants of the avian neural tube7-9. In vitro isolation of NC cells facilitates the study of NC dynamics including proliferation, migration, and multipotency. Further work in the avian and rat systems demonstrated that explanted NC cells retain their NC potential when transplanted back into the embryo10-13. Because these inherent cellular properties are preserved in explanted NC progenitors, the neural tube explant assay provides an attractive option for studying the NC in vitro. 
To attain a better understanding of the mammalian NC, many methods have been employed to isolate NC populations. NC-derived progenitors can be cultured from post-migratory locations in both the embryo and adult to study the dynamics of post-migratory NC progenitors11,14-20, however isolation of NC progenitors as they emigrate from the neural tube provides optimal preservation of NC cell potential and migratory properties13,21,22. Some protocols employ fluorescence activated cell sorting (FACS) to isolate a NC population enriched for particular progenitors11,13,14,17. However, when starting with early stage embryos, cell numbers adequate for analyses are difficult to obtain with FACS, complicating the isolation of early NC populations from individual embryos. Here, we describe an approach that does not rely on FACS and results in an approximately 96% pure NC population based on a Wnt1-Cre activated lineage reporter23. 
The method presented here is adapted from protocols optimized for the culture of rat NC11,13. The advantages of this protocol compared to previous methods are that 1) the cells are not grown on a feeder layer, 2) FACS is not required to obtain a relatively pure NC population, 3) premigratory NC cells are isolated and 4) results are easily quantified. Furthermore, this protocol can be used for isolation of NC from any mutant mouse model, facilitating the study of NC characteristics with different genetic manipulations. The limitation of this approach is that the NC is removed from the context of the embryo, which is known to influence the survival, migration and differentiation of the NC2,24-28.

Isolation of embryonic neural crest from the neural tube facilitates the use of in vitro methods for studying migration, self-renewal, and multipotency of neural crest.

胎児マウス神経管から神経堤細胞の単離および培養

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to ...

Dissection, Culture, and Analysis of Xenopus laevis Embryonic Retinal Tissue

The process by which the anterior region of the neural plate gives rise to the vertebrate retina continues to be a major focus of both clinical and basic research. In addition to the obvious medical relevance for understanding and treating retinal disease, the development of the vertebrate retina continues to serve as an important and elegant model system for understanding neuronal cell type determination and differentiation1-16. The neural retina consists of six discrete cell types (ganglion, amacrine, horizontal, photoreceptors, bipolar cells, and M&uuml;ller glial cells) arranged in stereotypical layers, a pattern that is largely conserved among all vertebrates 12,14-18.
While studying the retina in the intact developing embryo is clearly required for understanding how this complex organ develops from a protrusion of the forebrain into a layered structure, there are many questions that benefit from employing approaches using primary cell culture of presumptive retinal cells 7,19-23. For example, analyzing cells from tissues removed and dissociated at different stages allows one to discern the state of specification of individual cells at different developmental stages, that is, the fate of the cells in the absence of interactions with neighboring tissues 8,19-22,24-33. Primary cell culture also allows the investigator to treat the culture with specific reagents and analyze the results on a single cell level 5,8,21,24,27-30,33-39. Xenopus laevis, a classic model system for the study of early neural development 19,27,29,31-32,40-42, serves as a particularly suitable system for retinal primary cell culture 10,38,43-45.
Presumptive retinal tissue is accessible from the earliest stages of development, immediately following neural induction 25,38,43. In addition, given that each cell in the embryo contains a supply of yolk, retinal cells can be cultured in a very simple defined media consisting of a buffered salt solution, thus removing the confounding effects of incubation or other sera-based products 10,24,44-45.
However, the isolation of the retinal tissue from surrounding tissues and the subsequent processing is challenging. Here, we present a method for the dissection and dissociation of retinal cells in Xenopus laevis that will be used to prepare primary cell cultures that will, in turn, be analyzed for calcium activity and gene expression at the resolution of single cells. While the topic presented in this paper is the analysis of spontaneous calcium transients, the technique is broadly applicable to a wide array of research questions and approaches (Figure 1).

Dissection, Culture, and Analysis of Xenopus laevis Embryonic Retinal Tissue

Xenopus laevis provides an ideal model system for studying cell fate specification and physiological function of individual retinal cells in primary cell culture. Here we present a technique for dissecting retinal tissues and generating primary cell cultures that are imaged for calcium activity and analyzed by in situ hybridization.

の解離、文化、および分析<em&gt;アフリカツメガエル</em&gt;胚性網膜組織

The process by which  the anterior region of the neural plate gives rise to the vertebrate retina  continues to be a major focus of both clinical and ...

Simultaneous Pre- and Post-synaptic Electrophysiological Recording from Xenopus Nerve-muscle Co-cultures

Much information about the coupling of presynaptic ionic currents with the release of neurotransmitter has been obtained from invertebrate preparations, most notably the squid giant synapse1. However, except for the preparation described here, few vertebrate preparations exist in which it is possible to make simultaneous measurements of neurotransmitter release and presynaptic ionic currents. Embryonic Xenopus motoneurons and muscle cells can be grown together in simple culture medium at room temperature; they will form functional synapses within twelve to twenty-four hours, and can be used to study nerve and muscle cell development and synaptic interactions for several days (until overgrowth occurs). Some advantages of these co-cultures over other vertebrate preparations include the simplicity of preparation, the ability to maintain the cultures and work at room temperature, and the ready accessibility of the synapses formed2-4. The preparation has been used widely to study the biophysical properties of presynaptic ion channels and the regulation of transmitter release5-8. In addition, the preparation has lent itself to other uses including the study of neurite outgrowth and synaptogenesis9-12, molecular mechanisms of neurotransmitter release13-15, the role of diffusible messengers in neuromodulation16,17, and in vitro synaptic plasticity18-19.

Simultaneous Pre- and Post-synaptic Electrophysiological Recording from Xenopus Nerve-muscle Co-cultures

This video demonstrates the procedures used to grow primary cultures of embryonic Xenopus nerve and muscle cells and the usefulness of this preparation for making simultaneous pre- and post-synaptic patch clamp recordings.

からの同時プリとポストシナプスの電気生理学的記録<em&gt;アフリカツメガエル</em&gt;神経筋共培養

Much information about the coupling of  presynaptic ionic currents with the release of neurotransmitter has been obtained  from invertebrate preparations, ...

One Mouse, Two Cultures: Isolation and Culture of Adult Neural Stem Cells from the Two Neurogenic Zones of Individual Mice

The neurosphere assay and the adherent monolayer culture system are valuable tools to determine the potential (proliferation or differentiation) of adult neural stem cells in vitro. These assays can be used to compare the precursor potential of cells isolated from genetically different or differentially treated animals&#160;to determine the effects of exogenous factors on neural precursor cell proliferation and differentiation and to generate neural precursor cell lines that can be assayed over continuous passages. The neurosphere assay is traditionally used for the post-hoc identification of stem cells, primarily due to the lack of definitive markers with which they can be isolated from primary tissue&#160;and has the major advantage of giving a quick estimate of precursor cell numbers in brain tissue derived from individual animals. Adherent monolayer cultures, in contrast, are not traditionally used to compare proliferation between individual animals, as each culture is generally initiated from the combined tissue of between 5-8 animals. However, they have the major advantage that, unlike neurospheres, they consist of a mostly homogeneous population of precursor cells and are useful for following the differentiation process in single cells. Here, we describe, in detail, the generation of neurosphere cultures and, for the first time, adherent cultures from individual animals. This has many important implications including paired analysis of proliferation and/or differentiation potential in both the subventricular zone (SVZ) and dentate gyrus (DG) of treated or genetically different mouse lines, as well as a significant reduction in animal usage.

Here we describe a detailed protocol for the simultaneous generation of neural precursor cell cultures, as either adherent monolayers or neurospheres, from the subventricular zone and dentate gyrus of individual adult mice.

マウスを一回、二つの文化：個々のマウスの二つの神経原性のゾーンから成体神経幹細胞の単離および培養

The neurosphere assay and the adherent monolayer culture system are valuable tools to determine the potential (proliferation or differentiation) of adult ...

A Simple Behavioral Assay for Testing Visual Function in Xenopus laevis

Measurement of the visual function in the tadpoles of the frog, Xenopus laevis, allows screening for blindness in live animals. The optokinetic response is a vision-based, reflexive behavior that has been observed in all vertebrates tested. Tadpole eyes are small so the tail flip response was used as alternative measure, which requires a trained technician to record the subtle response. We developed an alternative behavior assay based on the fact that tadpoles prefer to swim on the white side of a tank when placed in a tank with both black and white sides. The assay presented here is an inexpensive, simple alternative that creates a response that is easily measured. The setup consists of a tripod, webcam and nested testing tanks, readily available in most Xenopus laboratories. This article includes a movie showing the behavior of tadpoles, before and after severing the optic nerve. In order to test the function of one eye, we also include representative results of a tadpole in which each eye underwent retinal axotomy on consecutive days. Future studies could develop an automated version of this assay for testing the vision of many tadpoles at once.

A Simple Behavioral Assay for Testing Visual Function in Xenopus laevis

Xenopus laevis tadpoles prefer swimming on the white side of a black/white tank. This behavior is guided by their vision. Based on this behavior, we present a simple assay to test the visual function of tadpoles.

テスト視機能のための簡単​​な行動アッセイ<em&gt;アフリカツメガエル</em

Measurement of the visual function in the tadpoles of the frog, Xenopus laevis, allows screening for blindness in live animals. The optokinetic response ...

Recording Temperature-induced Neuronal Activity through Monitoring Calcium Changes in the Olfactory Bulb of Xenopus laevis

The olfactory system, specialized in the detection, integration and processing of chemical molecules is likely the most thoroughly studied sensory system. However, there is piling evidence that olfaction is not solely limited to chemical sensitivity, but also includes temperature sensitivity. Premetamorphic Xenopus laevis are translucent animals, with protruding nasal cavities deprived of the cribriform plate separating the nose and the olfactory bulb. These characteristics make them well suited for studying olfaction, and particularly thermosensitivity. The present article describes the complete procedure for measuring temperature responses in the olfactory bulb of X. laevis larvae. Firstly, the electroporation of olfactory receptor neurons (ORNs) is performed with spectrally distinct dyes loaded into the nasal cavities in order to stain their axon terminals in the bulbar neuropil. The differential staining between left and right receptor neurons serves to identify the &#947;-glomerulus as the only structure innervated by contralateral presynaptic afferents. Secondly, the electroporation is combined with focal bolus loading in the olfactory bulb in order to stain mitral cells and their dendrites. The 3D brain volume is then scanned under line-illumination microscopy for the acquisition of fast calcium imaging data while small temperature drops are induced at the olfactory epithelium. Lastly, the post-acquisition analysis allows the morphological reconstruction of the thermosensitive network comprising the &#947;-glomerulus and its innervating mitral cells, based on specific temperature-induced Ca2+ traces. Using chemical odorants as stimuli in addition to temperature jumps enables the comparison between thermosensitive and chemosensitive networks in the olfactory bulb.

Recording Temperature-induced Neuronal Activity through Monitoring Calcium Changes in the Olfactory Bulb of Xenopus laevis

Here we describe a protocol for measuring and analyzing temperature responses in the olfactory bulb of Xenopus laevis. Olfactory receptor neurons and mitral cells are differentially stained, after which calcium changes are recorded, reflecting a sensitivity of some neural networks in the bulb to temperature drops induced at the nose.

の嗅球にカルシウム変化をモニタリングを通じて温度によって誘発される神経活動を記録<em&gt;アフリカツメガエル</em

The olfactory system, specialized in the detection, integration and processing of chemical molecules is likely the most thoroughly studied sensory system. ...

Derivation of Leptomeninges Explant Cultures from Postmortem Human Brain Donors

Even though great progress has been made in the clinical characterization of Parkinson&#39;s disease, several studies report that the diagnosis of Parkinson&#39;s disease is not pathologically confirmed in up to 25% of clinically diagnosed Parkinson&#39;s disease. Therefore, tissue collected from clinically diagnosed patients with idiopathic Parkinson&#39;s disease can have a high rate of misdiagnosis; hence in vitro studies from such tissues to study Parkinson&#39;s disease as a preclinical model can become futile.
By collecting postmortem human leptomeninges with a confirmed neuropathological diagnosis of Parkinson&#39;s disease and characterized by nigrostriatal cell loss and intracellular protein inclusions called Lewy bodies, one can be certain that clinically observed parkinsonism is not caused by another underlying disease process (e.g. tumor, arteriosclerosis).
This protocol presents the dissection and preparation of postmortem human leptomeninges for derivation of a meningeal fibroblast culture. This procedure is robust and has a high success rate. The challenge of the culture is sterility as the brain procurement is generally not performed under sterile conditions. Therefore, it is important to supplement the culture media with a cocktail of penicillin, streptomycin, and amphotericin B.
The derivation of meningeal fibroblasts from autopsy-confirmed cases with Parkinson&#39;s disease provides the foundation for in vitro modeling of Parkinson&#39;s disease. Meningeal fibroblasts appear 3-9 days after sample preparation and about 20-30 million cells can be cryopreserved in 6-8 weeks. The meningeal fibroblast culture is homogenous and the cells express fibronectin, a commonly used marker to identify meninges.

The leptomeninges explant culture protocol from human postmortem brain is a technically robust and simple way to derive fibronectin-positive meningeal fibroblasts within 6-8 weeks and cryopreserve approximately 20-30 million cells.

死後ヒト脳ドナーから軟膜植片培養物の導出

Even though great progress has been made in the clinical characterization of Parkinson&#39;s disease, several studies report that the diagnosis of ...

Time-dependent Increase in the Network Response to the Stimulation of Neuronal Cell Cultures on Micro-electrode Arrays

Micro-electrode arrays (MEAs) can be used to investigate drug toxicity, design paradigms for next-generation personalized medicine, and study network dynamics in neuronal cultures. In contrast with more traditional methods, such as patch-clamping, which can only record activity from a single cell, MEAs can record simultaneously from multiple sites in a network, without requiring the arduous task of placing each electrode individually. Moreover, numerous control and stimulation configurations can be easily applied within the same experimental setup, allowing for a broad range of dynamics to be explored. One of the key dynamics of interest in these in vitro studies has been the extent to which cultured networks display properties indicative of learning. Mouse neuronal cells cultured on MEAs display an increase in response following training induced by electrical stimulation. This protocol demonstrates how to culture neuronal cells on MEAs; successfully record from over 95% of the plated dishes; establish a protocol to train the networks to respond to patterns of stimulation; and sort, plot, and interpret the results from such experiments. The use of a proprietary system for stimulating and recording neuronal cultures is demonstrated. Software packages are also used to sort neuronal units. A custom-designed graphical user interface is used to visualize post-stimulus time histograms, inter-burst intervals, and burst duration, as well as to compare the cellular response to stimulation before and after a training protocol. Finally, representative results and future directions of this research effort are discussed.

Mouse neuronal cells cultured on multi-electrode arrays display an increase in response following electrical stimulation. This protocol demonstrates how to culture neurons, how to record activity, and how to establish a protocol to train the networks to respond to patterns of stimulation.

マイクロ電極アレイにおけるニューロン細胞培養の刺激に対するネットワーク応答の時間依存的増加

Micro-electrode arrays (MEAs) can be used to investigate drug toxicity, design paradigms for next-generation personalized medicine, and study network ...

Simultaneous Recordings of Cortical Local Field Potentials, Electrocardiogram, Electromyogram, and Breathing Rhythm from a Freely Moving Rat

Monitoring the physiological dynamics of the brain and peripheral tissues is necessary for addressing a number of questions about how the brain controls body functions and internal organ rhythms when animals are exposed to emotional challenges and changes in their living environments. In general experiments, signals from different organs, such as the brain and the heart, are recorded by independent recording systems that require multiple recording devices and different procedures for processing the data files. This study describes a new method that can simultaneously monitor electrical biosignals, including tens of local field potentials in multiple brain regions, electrocardiograms that represent the cardiac rhythm, electromyograms that represent awake/sleep-related muscle contraction, and breathing signals, in a freely moving rat. The recording configuration of this method is based on a conventional micro-drive array for cortical local field potential recordings in which tens of electrodes are accommodated, and the signals obtained from these electrodes are integrated into a single electrical board mounted on the animal's head. Here, this recording system was improved so that signals from the peripheral organs are also transferred to an electrical interface board. In a single surgery, electrodes are first separately implanted into the appropriate body parts and the target brain areas. The open ends of all of these electrodes are then soldered to individual channels of the electrical board above the animal's head so that all of the signals can be integrated into the single electrical board. Connecting this board to a recording device allows for the collection of all of the signals into a single device, which reduces experimental costs and simplifies data processing, because all data can be handled in the same data file. This technique will aid the understanding of the neurophysiological correlates of the associations between central and peripheral organs.

This study introduces a method for the simultaneous recording of local field potentials in the brain, electrocardiograms, electromyograms, and breathing signals of a freely moving rat. This technique, which reduces experimental costs and simplifies data analysis, will contribute to the understanding of the interactions between the brain and peripheral organs.

皮質局所電界電位、心電図、筋電図、呼吸リズム自由行動ラットからの同時録音

Monitoring the physiological dynamics of the brain and peripheral tissues is necessary for addressing a number of questions about how the brain controls ...

Methods Article

からの神経外植文化